Monotopic topology is required for lipid droplet targeting of ancient ubiquitous protein 1

Abstract

proteins of the LD surface need unconventional targeting mechanisms ( 3 ). Previous studies on targeting of proteins to LDs have pointed to the importance of hydrophobic stretches or amphipatic domains in soluble proteins ( 4-12 ). For integral proteins, hydrophobic domains, often located at the end of the polypeptide chain (13)(14)(15)(16)(17)(18)(19), and charged residues ( 20 ) have been found to be essential for droplet targeting. On theoretical grounds, we have proposed that targeting of integral proteins to LDs requires a hairpin-like monotopic topology ( 21 ), but experimental evidence for a direct connection between topology and targeting is lacking so far. Additional complexity comes from the fact that many integral proteins of LDs exhibit additional localizations, sometimes to the plasma membrane ( 22-27 ), but mostly to the endoplasmic reticulum (ER) ( 28-34 ). One such protein is ancient ubiquitous protein 1 (AUP1) that specifi cally localizes to . AUP1 is a 410 amino acids long, integral protein with a single predicted membrane domain ( 36, 37 ). The membrane domain of AUP1 is located internally and both the N and C termini of AUP1 are facing the cytoplasm, resulting in monotopic/hairpin topology ( 36 ).AUP1 has multiple functional domains and is reported to facilitate ER-associated protein degradation, inside-out signaling in platelets, and neutral lipid storage ( 35-39 ). Domains important for a function of AUP1 in ER-associated degradation are found in its C-terminal region. The CUE (coupling of ubiquitin to ER degradation) domain binds dislocation substrates and components of the ER quality control machinery ( 37 ), and the G2 binding region (G2BR) domain recruits a specifi c E2 ubiquitin conjugase, Ube2g2 ( 36 ). The function of the N-terminal region of AUP1 is lesswell understood. It contains the membrane domain and a predicted acyltransferase domain, which was proposed to Abstract Ancient ubiquitous protein 1 (AUP1) is a multifunctional protein, which acts on both lipid droplets (LDs) and the endoplasmic reticulum (ER) membrane. Double localization to these two organelles, featuring very different membrane characteristics, was observed also for several other integral proteins, but little is known about the signals and mechanisms behind dual protein targeting to ER and LDs. Here we dissect the AUP1 targeting signals by analyses of localization and topology of several deletion and point mutants. We found that AUP1 is inserted into the membrane of the ER in a monotopic hairpin fashion, and subsequently transported to the hemi-membrane of LDs. A single domain localized in the N-terminal part of AUP1 enables its ER residence, the monotopic insertion, and the LD localization. Different specifi c residues within this multifunctional domain are responsible for achieving the complex spatial distribution pattern. A mutation of three amino acids, which changes AUP1 topology from hairpin to transmembrane, abolishes LD localization. These fi ndings suggest that the cell is able to target a protein to mult...

Establishing the lipid droplet proteome: Mechanisms of lipid droplet protein targeting and degradation

“…Examples of Class I hairpin proteins that traffic from the ER to LDs include UBXD8 [58,59], AUP1 [60], caveolin-1 [61,62], DGAT2 [63], and GPAT4 [40]. A common feature of many of these hairpin domains is a conserved proline residue positioned in the center of the hydrophobic region.…”

“…The hydrophobic regions of these proteins are at their extreme N-termini, raising the possibility that the position of the hydrophobic region determines the structure that these regions adopt as well as the importance of the proline. It should be noted that while the hydrophobic region of Class I LD proteins is generally necessary and sufficient for LD targeting, trafficking of some proteins from the ER to LDs is impacted by flanking positively-charged sequences [60,62]. The significance of these regions is unclear, but they may mediate interactions with specific negatively charged lipid headgroups or proteins in the LD monolayer.…”

Lipid droplets and steroidogenic cells

“…For mediating cholesterol homeostasis, Aup1, which has a single domain that allows for its insertion into the ER as well as into LDs (57), was shown to interact with ERAD and facilitate binding of gp78 and Trc8 to ubiquitin-conjugating enzyme (Ubc7) at the LD surface (54). UBX-domain containing protein (Ubx2), which is an ER protein and selectively transports misfolded proteins for ERAD, was shown to be crucial in LD maintenance, with its deletion leading to a 50% decrease in intracellular TAG accumulation (58).…”

Molecular Mechanisms and Physiological Significance of Organelle Interactions and Cooperation

“…A subset of proteins traffic to droplets via the ER, as covered in a recent review (Walther and Farese, 2012 (Stevanovic and Thiele, 2013). Another line of evidence is in systems in which droplet formation is stimulated, by incubation of cells in oleic acid or induction of a neutral lipidsynthesizing enzyme.…”

“…These include derlin-1, UBXD2, UBXD8, p97/VCP, and AUP1 (Suzuki et al, 2012;Olzmann et al, 2013;Stevanovic and Thiele, 2013). Evidence for colocalization is their appearance in proteomes of isolated droplets, and live and fixed cell fluorescence with antibodies or tagged proteins.…”